Structural insight for substrate tolerance to 2-deoxyribose-5-phosphate aldolase from the pathogen Streptococcus suis

Abstract

2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/β)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and β8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility.

Conformational Sampling of the Intrinsically Disordered C-Terminal Tail of DERA Is Important for Enzyme Catalysis

“…In all the available crystal structures of DERAs containing a C-terminal tyrosine, 24 , 32 , 33 , 35 , 36 , 74 the electron density for the C-terminal tail is poorly defined, which prevents understanding the structural and functional basis of the participation of Y259 in catalysis. To characterize the dynamics of the C-terminal tail in solution, we acquired { 1 H} 15 N heteronuclear NOE (hetNOE) values for DERAm.…”

“… 23 Therefore, DERA is considered as a promising pharmaceutical target against human bacterial pathogens. 24 , 25 In addition to this, DERA is unique in catalyzing the cross-aldol condensation between two aldehydes and is utilized as an environmentally friendly biocatalyst for the synthesis of drugs such as statins. 26 − 28 However, the absence of a phosphate group from the substrate severely curtails the catalytic efficiency of DERA, 29 thereby restricting its use as an efficient biocatalyst to phosphorylated aldehydes.…”

“…In vivo, DERA catalyzes the reversible conversion of acetaldehyde and glyceraldehyde-3-phosphate (G3P) to generate deoxyribose-5-phosphate (dR5P) , and provides key intermediates essential for different metabolic pathways, e.g., glycolysis, the pentose-phosphate pathway, and nucleotide catabolism . Therefore, DERA is considered as a promising pharmaceutical target against human bacterial pathogens. , In addition to this, DERA is unique in catalyzing the cross-aldol condensation between two aldehydes and is utilized as an environmentally friendly biocatalyst for the synthesis of drugs such as statins. − However, the absence of a phosphate group from the substrate severely curtails the catalytic efficiency of DERA, thereby restricting its use as an efficient biocatalyst to phosphorylated aldehydes. Additionally, mutations of residues interacting with the dR5P phosphate group have also been shown to drastically decrease the activity of ec DERA .…”

“…Despite several biochemical studies suggesting that the C-terminal tyrosine (i.e., Y259) is crucial for efficient catalysis of dR5P breakdown, , the absence of electron density for the last eight C-terminal residues (residues D252–Y259 in ec DERA: i.e., the C-terminal tail) in all available crystal structures of DERAs containing a C-terminal tyrosine (including ec DERA) ,,,, has precluded acquiring insights into the role of the tyrosine as well as the C-terminal tail. As observed in ec DERA, the C-terminal tyrosine residue of class I rabbit muscle fructose-1,6-disphosphate aldolase (FBPA) is important for the catalysis of dihydroxyacetone phosphate (DHAP) breakdown.…”

Synthetic Activity of Recombinant Whole Cell Biocatalysts Containing 2‐Deoxy‐D‐ribose‐5‐phosphate Aldolase from Pectobacterium atrosepticum

“…Structural studies have shown that the wide substrate tolerance range of DERA is related to the C-terminus which is shorter and more flexible than that of other classes of aldolases. [32] This C-terminal tail generates an equilibrium between an open and a closed state. In the closed state, the C-terminal tyrosine enters to the active site to participate in a catalytic deprotonation step, while the open state allows substrate binding and product release.…”